1. Field of the Invention
The present invention relates to a method for producing a cloned dog. More particularly, the present invention relates to a method for producing a cloned dog by enucleating an oocyte of a dog to prepare an enucleated oocyte, transferring a somatic cell of the dog into the enucleated oocyte and electrofusing it under optimized conditions to prepare a nuclear transfer embryo, and transferring the nuclear transfer embryo to an oviduct of its surrogate mother.
2. Description of the Related Art
With the recent development of somatic cell nuclear transfer technology by cell fusion or intracytoplasmic cell injection, animals are actually being cloned.
The somatic cell nuclear transfer technology, which allows living offspring to be born without undergoing meiosis and haploid germ cell which generally occur in a generative process, is a method of developing new individuals by transferring diploid somatic cells of adults into enucleated cells to produce embryos and transferring the embryos in vivo. Generally, in somatic cell nuclear transfer technology, recipient oocytes of somatic cell donor nuclei are used after they are artificially cultured in vitro to metaphase II of meiosis. Then, in order to prevent the development of chromosomal abnormality resulting from the somatic cell nuclear transfer, the mature oocytes are enucleated before transferring the somatic cells. After transferring the somatic cells into the perivitelline space or cytoplasm of the mature oocytes, the enucleated oocytes and the somatic cells are physically fused with each other by electrical stimulation. The fused enucleated oocytes and the somatic cells are activated by electrical stimulation or chemical substances and transferred into surrogate mothers to produce living offspring.
Such somatic cell nuclear transfer technology can be widely used in the field, for example, in the propagation of superior animals, conservation of rare or nearly extinct animals, production of certain nutrients, production of therapeutic bio-materials, production of animals for organ transplantation, production of animals with diseases or disorders, and production of medically worthy animals for alternative treatments to organ transplantation such as gene therapy.
Animal cloning was first accomplished by Dr. Wilmut of the Roslin Institute, England, by taking a mammary gland cell from a six-year old sheep, transferring the cell into an enucleated oocyte to prepare a nuclear transfer embryo, and transferring the embryo in vivo, thereby producing a cloned animal named Dolly. Thereafter, it was reported that cloned cows, mice, goats, pigs and rabbits were produced by nuclear transfer using somatic cells (see WO 9937143 A2, EP 930009A1, WO 9934669A1, WO 9901164A1 and U.S. Pat. No. 5,945,577).
Meanwhile, not only cloning of farm animals such as cows and pigs, but recent cloning of pet animals such as dogs has also attracted a great deal of interest. A cat was the first pet animal to be cloned, and a study on dog cloning was conducted with several millions of dollars in funding.
However, it is very difficult to implement cloning by somatic cell nuclear transfer due to species-specific generative properties of the dog. The present inventors produced a cloned dog for the first time, but many difficulties remained in putting such cloning into practical use because the cloning efficiency was too low.
Accordingly, while researching an improved method for producing a cloned dog using somatic cell nuclear transfer, the present inventors have established optimized electrofusion conditions of the enucleated oocyte and the nuclear donor cell, produced a nuclear transfer embryo, and transferred the nuclear transfer embryo into a surrogate mother to produce a cloned dog with high efficiency, thereby completing the present invention.